C57bl 6 male and female mice

C57BL/6 male and female mice (8 weeks old, Charles River, USA) were anaesthetized using isoflurane gas and the right forelimb was shaved and swabbed with povidone-iodine. Mice received a dose of buprenorphine and carprofen for pain relief. An incision was performed along the right forearm and the soft tissue over the radius was blunt-dissected using a periosteal elevator to expose the bone. A 2.5 ​mm defect was created in the center of the radius using a custom-made parallel double-bladed bone cutter. Care was taken to leave the ulna intact, and the 4 ​mm length implant was introduced into the radial bone defect, abutting its proximal and distal ends. After repositioning the muscle and skin, the incision was closed with a degradable suture and the mice were monitored for signs of distress, movement, and weight loss. 6 mice (3 males and 3 females) were used per experimental condition.

All experiments were conducted in accordance with protocols approved by the local ethics committee for animal experiments in Sweden (Stockholm North Animal Ethics Board) and the United States (the Institutional Animal Care and Use Committee of the University of Texas at Dallas), and were in accordance with the International Association for the Study of Pain guidelines. C57BL/6 male and female mice (10-12 weeks, 20-25 g) were purchased from Charles River Laboratories (Freiberg, Germany) and Janvier Labs (Le Genest-Saint-Isle, France). Myeloid cell-specific TLR4 depleted male and female mice (LysM-TLR4^fl/fl) were generated as previously described.^{25 (link)} In brief, TLR^fl/fl mice were crossed with mice expressing Cre under LysM promoter. The resulting LysM-TLR4^fl/fl and TLR4^fl/fl (control mice) were backcrossed 8 generations to a C57BL/6 background at the University of Texas at Dallas. Wild-type (PAR2^+/+) and proteinase-activated receptor 2 (PAR2)-deficient (PAR2^−/−) male and female mice were obtained from Jackson Laboratory (Bar Harbor, ME) and bred at University of Texas at Dallas. Animals were housed in a pathogen-free environment with 5 mice per cage with water and food ad libitum in animal facilities at Karolinska Institutet and University of Texas at Dallas in a pathogen-free environment with standard temperature and 12-hour light/dark cycle.